Considerable effort has been exerted to identify markers that would be useful in assessing brain damage, such as that caused by stroke or Alzheimer's disease. Early diagnosis of, for example, ischemic stroke, is believed critical in order to permit an appropriate intervention, such as administration of recombinant tissue plasminogen activator which has been shown to be highly effective, if administered early, in reducing mortality and morbidity resulting from stroke. In addition, other forms of brain damage, such as hemorrhagic stroke, damage to asphyxiated term infants, brain damage resulting from cardiac surgery, Alzheimer's or miscellaneous neurodegenerative disorders are desired to be assessed; although additional diagnostic procedures may be required to distinguish among these various possibilities in some cases.
It is generally recognized that brain damage of various types can be indicated by the presence in fluids such as cerebrospinal fluid (CSF) or more conveniently, in serum or plasma or urine, of proteins or other substances that are generally characteristic of the brain. The desirability of identifying such factors that could be used for diagnoses so as to identify appropriate treatment or simply for prognosis has also been recognized widely. See, for example, Warlow, C., Lancet (2003) 362:1211-1224; Qureshi, A., et al., New Eng J Med, (2001) 344:1450-1460; Marler, J. R., et al., Science (2003) 301:157; Garca-Alix, A., et al., Acta Paediatr (2001) 90:1103-1105; Verbeek, M. M., et al., Ann Clin Biochem. (2003) 40:25-40.
Because of this understanding, various groups have undertaken proteomic studies of the brain to identify characteristic brain proteins. An analysis of the brain proteins in mice using 2-D electrophoresis and mass spectrometry was published by Gauss, C., et. al., Electrophoresis (1999) 20:575-600. The pattern showed 8,767 protein spots of which 200 were identified in the article. Two-dimensional gel electrophoresis and mass spectroscopy has been applied to CSF obtained six hours postmortem and compared to fresh CSF. Thirteen candidate proteins, some of which had been previously associated with neurodegenerative diseases were identified (Lescuyer, et al. Proteomics (2004) 4:2234-2241). A general analysis of this approach is described by Lubec, G., et al., Progress in Neurobiol (2003) 611:1-19. A news story by Abbott, A., in Nature (2003) 425:110 points out that while analysis of human brains has to rely on autopsied tissue, mouse brains can be analyzed at various ages using fresh tissue. Attempts have also been made to analyze genomic influences on stroke or other brain damage-associated conditions by Kato, N., et al., Atherosclerosis (2002) 163:279-286 and Rosand, J., et al., Stroke (2003) 34:2512-2517, for example.
There are a number of biomarkers of brain injury that have been reported in the scientific literature. These include 5-100B, neuron-specific enolase (NSE), glial fibrillary associated protein (GFAP), myelin basic protein (MBP) and others. (Aurell, A., et al., Stroke (1991) 22:1254-1258; Barone, F. C., et al., Brain Res (1993) 623:77-82; Cunningham, R. T., et al., Eur J Clin Invest (1991) 21:497-500; Hardemark, H. G., et al., J Neurosurg (1989) 71:727-731; Hardemark, H. G., et al., Stroke (1988) 19:1140-1144; Hatfield, R. H., et al., Brain Res (1992) 577:249-252; Hay, E., et al., J Neurol Neurosurg Psychiatry (1984) 47:724-729; Noppe, M., et al., Clin Chim Acta (1986) 155:143-150; Steinberg, R., et al., J Neurochem (1984) 43:19-24).
S-100B is a Ca2+-binding protein that modulates complex neuronal-glial interactions and is found mostly in glia, melanocytes, Schwann cells, Langerhans cells and anterior pituitary cells, but not in neurons. Elevated serum levels of S-100B have been associated with stroke, post-cardiac arrest brain injury and traumatic head injury. (Aurell, A, et al., Stroke (1991) 22:1254-1258; Hardemark, H. G., et al., J Neurosurg (1989) 71:727-731; Noppe, M, et al., Clin Chim Acta (1986) 155:143-150; Bottiger, B. W., et al., Circulation (2001) 103:2694-2698, Sellman, M., et al., Scand J. Thor. Cardiovasc. Surg. (1992) 26:39-45, Shaabam, A., et al., Brit J Anesthesia (2000) 85:287-298).
Leviton, A., et al., Acta Paediatr (2002) 91:9-13 further studied the use of S-100B, glial fibrillary acidic protein (GFAP) and NSE as markers for brain damage in children with the view to their diagnostic capability to assess such injury. Rothoerl, R. D., et al., Acta Neurochem (2000) Suppl. 76:97-100 showed that the serum level of S-100B is also elevated after severe head injury; Abraha, H. D., et al., Ann Clin Biochem. (1997) 34:546-550 suggest that measurement of serum S-100 protein is a useful prognostic marker of clinical outcome in acute stroke. Further confirmation that S-100B and NSE are significant markers of brain damage is set forth in Mussack, T., et al., Shock (2002) 18:395-400 and in a comment on this article by Vos, P. F. et al., ibid. 481-482. It is noted that increased serum concentrations of S-100B, GFAP, and NSE have been associated with various acute central nervous system disorders.
However, S-100B is not brain specific (Vaage, J., et al., J Thorac Cardiovasc Surg (2001) 122:853-855; Unden, J., Scand J Infect Dis (2004) 36:10-13) since it is also expressed in white and brown adipose tissue, skin, skeletal muscle, melanoma and glioblastoma cells (Zimmer, D. B., et al., Brain Res Bull (1995) 37:417-429; Ilg, E. C., et al., Int J Cancer (1996) 68:325-332), as well as in muscle, heart and the kidneys (Baudier, J, et al., J Biol Chem (1986) 261:8192-8203; Missler, U., et al., Eur J Clin Chem Clin Biochem (1995) 33:743-748).
NSE represents the gamma, gamma-dimer of the protein enolase (2-phospho-D-glycerate hydrolase), which is a soluble enzyme of the glycolytic pathway with a total molecular weight of approximately 80 kDa (Schmechel, D., et al., Science (1978) 199:313-315). NSE is expressed in neuronal cytoplasm and dendrites and in cells of the amine precursor uptake and decarboxylation (APUD) cell system. Early clinical studies are available demonstrating elevated serum NSE titers in stroke or cardiac arrest patients (Persson, L., et al., Stroke (1987) 18:911-918; Dauberschmidt, R., et al., Mol Chem Neuropathol (1991) 14:237-245; Schaarschmidt, H, et al., Stroke (1994) 25:558-565. In addition, tumor cells in APUDomas, neuroblastomas, seminomas, and small-cell carcinoma of the lung also express NSE. For this reason, NSE has been studied as a diagnostic and prognostic serum marker in clinical management of such neoplasms. However, NSE can also be found in red cells and platelets and cannot be considered specific for brain (Johnsson, P. J., Cardiothorac Vasc Anesth (1996) 10:120-126).
Combinations of markers have been used in an attempt to obtain better sensitivity and specificity for stroke. One group has utilized the combination of brain markers neuron-specific enolase, myelin basic protein, and S-100B (Kupchak, P., et al., Clin Chem (2005) 51(6):A119 and A120; abstracts). Another group evaluated>50 protein biomarkers and chose S-100B, B-type neurotrophic growth factor, Von Willebrand factor, matrix metalloproteinase-9 and monocyte chemotactic protein-1 (Reynolds, M., Clin Chem (2003) 45(10):1733-1739). In another study, biomarkers based on brain damage (S100B), inflammation (matrix metalloproteinase-9 and vascular cell adhesion molecule) and thrombosis (Von Willebrand factor) were combined to identify acute stroke (Lynch, et al., Stroke (2004) 35:57-63).
An application has been submitted to FDA for a multimarker diagnostic device for acute stroke by Biosite, Inc. The markers are 5-100B, brain natriuretic protein, D-dimer, and matrix metalloproteinase-9.
At present, there is a need for additional and more reliable markers of brain damage than those currently available, even if combined with markers of phenomena other than brain injury. As noted above, all currently utilized brain damage markers are not sufficiently specific.